The Mixed Lineage Leukemia (MLL) gene codes for a histone methylase, which is frequently mutated by chromosomal translocations in human leukemias associated with a poor clinical outcome. The studies proposed in this application address the hypothesis that the leukemogenic actions of MLL oncoproteins are critically dependent on proteins recently discovered to associate with MLL or its fusion partners, and that the activities or interactions of these associated factors constitute potential targets for molecular therapies. This hypothesis is based on substantial preliminary studies that have resulted in the purification of multi-protein complexes containing MLL or its major fusion partners, and identification of proteins that associate with wild type and/or mutant MLL proteins. One of the recently identified MLL-associated proteins is menin, a product of the MEN1 tumor suppressor gene. Menin is an essential component of the MLL complex, is required for maintenance of Hox gene expression, and also interacts with oncogenic MLL fusion proteins. Studies in the first specific aim will determine the role of menin in the initiation and maintenance of MLL-mediated leukemogenesis using genetic approaches in pre-clinical and cell line transformation model systems. These studies will also establish the feasibility of targeting MLL-menin interactions as a molecular therapeutic strategy. Studies in the second aim will employ genetic and biochemical approaches to identify additional unknown factors that interact with transformation critical domains of MLL, establish their specific roles in leukemogenesis, and determine their feasibility as molecular therapeutic targets. Studies in the third specific aim are based on our discovery of an AF4 multi-protein complex that contains among other proteins the MLL fusion partner ENL. This novel complex links the two major families of MLL fusion partners on a common biochemical pathway. These observations will be extended by further characterizing the molecular nature of the AF4/ENL pathway, determining its implications for transcriptional regulation in general, and establishing its role in MLL-mediated leukemogenesis. The therapeutic value of targeting the activities or interactions of AF4 complex components with pathogenic roles in MLL leukemias will be interrogated using preclinical transformation models.